Sulforaphane, a secondary metabolite in crucifers, inhibits the oxidative stress adaptation and virulence of Xanthomonas by directly targeting OxyR

Abstract

Plant secondary metabolites perform numerous functions in the interactions between plants and pathogens. However, little is known about the precise mechanisms underlying their contribution to the direct inhibition of pathogen growth and virulence in planta. Here, we show that the secondary metabolite sulforaphane (SFN) in crucifers inhibits the growth, virulence, and ability of Xanthomonas species to adapt to oxidative stress, which is essential for the successful infection of host plants by phytopathogens. The transcription of oxidative stress detoxification-related genes (catalase [katA and katG] and alkylhydroperoxide-NADPH oxidoreductase subunit C [ahpC]) was substantially inhibited by SFN in Xanthomonas campestris pv. campestris (Xcc), and this phenomenon was most obvious in sax gene mutants sensitive to SFN. By performing microscale thermophoresis (MST) and electrophoretic mobility shift assay (EMSA), we observed that SFN directly bound to the virulence-related redox-sensing transcription factor OxyR and weakened the ability of OxyR to bind to the promoters of oxidative stress detoxification-related genes. Collectively, these results illustrate that SFN directly targets OxyR to inhibit the bacterial adaptation to oxidative stress, thereby decreasing bacterial virulence. Interestingly, this phenomenon occurs in multiple Xanthomonas species. This study provides novel insights into the molecular mechanisms by which SFN limits Xanthomonas adaptation to oxidative stress and virulence, and the findings will facilitate future studies on the use of SFN as a biopesticide to control Xanthomonas.

Keywords: OxyR; Xanthomonas species; oxidative stress; secondary metabolites; sulforaphane; virulence.

FIGURE 1

Sulforaphane (SFN) inhibits the growth of Xanthomonas spp. (a–c) Bacterial growth in NYG medium supplemented with 0 to 100 μM SFN. Xoo PXO99A, Xanthomonas oryzae pv. oryzae PXO99A; Xoc RS105, X. oryzae pv. oryzicola RS105; Xcc XC1, X. campestris pv. campestris XC1. Growth curves were monitored by measuring the OD₆₀₀ every 4 h after inoculation and all the samples were grown at 28°C until the stationary phase was achieved. Three biologically independent experiments were performed. The data in (a), (b) and (c) are presented as the mean ± SD (n = 3). (d–f) EC₅₀ of SFN that inhibits the growth of Xoo PXO99A, Xoc RS105, and Xcc XC1. Each data point is the average of three samples and error bars indicate SD (n = 3). Three biologically independent experiments were performed.

FIGURE 2

Identification of sax genes in Xanthomonas campestris pv. campestris. (a) Comparison of the genome regions of sax clusters between Pseudomonas syringae pv. tomato (Pst) DC3000 and Xanthomonas campestris pv. campestris (Xcc) XC1. Levels of sequence identity between the deduced proteins are shown. (b) Differential regulation of the transcription of sax genes in Xcc XC1 by 20 μM sulforaphane (SFN) after induction for 6 h. SFN‐mediated induction led to a greater than 10‐fold increase in the transcription of saxB and saxF in Xcc XC1. 16S rRNA was used as the endogenous control, the expression level in the wild‐type strain was assigned a numerical value of 1, and transcript levels were normalized to the 16S rRNA level. Experiments were performed three times with similar results. Each column indicates the mean of three biologically independent experiments. Vertical bars represent standard errors. The statistical analysis was performed using two‐way analysis of variance followed by Sidak's multiple comparison test, ***p < 0.001. (c) saxB and saxF are required for Xcc XC1 SFN resistance. Tolerance to SFN was determined by performing diffusion assays in which exponentially growing cells were spread on NYG agar plates containing 20 μl of SFN (20 mM) in a central hole. After 3 days of incubation, the inhibitory zone diameters of the cultures were measured and compared. Each dot represents the inhibitory zone diameter of a single biological replicate (n = 4). Experiments were performed three times with similar results. ***p < 0.001 (unpaired t test).

FIGURE 3

saxB and saxF of Xanthomonas campestris pv. campestris (Xcc) XC1 control virulence and antioxidant gene expression in planta. (a) Inactivation of ΔsaxB/F caused a reduction in virulence. Bacterial strains were inoculated into leaves of the host plant Brassica oleracea 'Jingfeng No. 1'. Lesion length was estimated 10 days after inoculation. (b) Lesion lengths of Xcc XC1 and ΔsaxB/F on the plants shown in (a). The virulence of the Xcc strains was tested by measuring the lesion length after inoculating Jingfeng No. 1 with bacteria. The values are presented as the means and standard deviations of triplicate measurements, each for 16 leaves. Asterisks indicate significant differences relative to the Xcc XC1 strain (unpaired t test, ***p < 0.001). (c) Inoculation with the ΔsaxB/F mutant led to greater reactive oxygen species (ROS) accumulation in Jingfeng No. 1 leaves than inoculation with Xcc XC1 or buffer (MgCl₂), as determined using 3,3′‐diaminobenzidine (DAB) staining. The DAB staining assay was performed as described in the experimental procedures. The DAB intensity was calculated from the digital photographs by determining the number of brown pixels. Average DAB measurements were calculated from 10 photographs of differently treated leaves from three independent experiments, and the statistical analysis was performed using one‐way analysis of variance followed by Tukey's multiple comparisons test (***p < 0.001, n = 10). (d) Mutation of saxB/F inhibited antioxidant gene expression in Xcc XC1 in planta. The transcript levels of the indicated genes in Xcc XC1 and ΔsaxB/F cells were determined in Jingfeng No. 1 using reverse transcription‐quantitative PCR 96 h after inoculation. 16S rRNA was used as the endogenous control, the expression level in the wild‐type strain was assigned a numerical value of 1, and transcript levels were normalized to the 16S rRNA level. Asterisks indicate significant differences relative to the Xcc Xc1 strain (multiple comparison t test, ***p < 0.001). The experiments were performed at least three times with similar results.

FIGURE 4

Analysis of antioxidant gene expression in sulforaphane (SFN)‐treated Xanthomonas campestris pv. campestris XC1 cells. (a) SFN inhibited XC1 antioxidant gene expression in vitro. XC1 cells were incubated with 100 μM SFN for 6 h and then the cultures were treated with 1 mM H₂O₂ for 10 min, after which samples were collected for the reverse transcription‐quantitative PCR (RT‐qPCR) assay. (b) SFN induced oxyR expression in XC1 in vitro. XC1 cells were incubated with 20 μM SFN for 6 h and oxyR transcript levels were determined using RT‐qPCR. 16S rRNA was used as the endogenous control, the expression level in the wild‐type strain was assigned a numerical value of 1, and transcript levels were normalized to the 16S rRNA level. The bars indicate the mean ± SD (n = 3) (a and b). Experiments were performed three times with similar results. The statistical analysis was performed using two‐way analysis of variance followed by Tukey's multiple comparisons test (***p < 0.001, *p < 0.05; n = 3).

FIGURE 5

Sulforaphane (SFN) inhibits the H₂O₂ resistance of Xanthomonas campestris pv. campestris XC1 growing in media. H₂O₂ tolerance was determined by performing diffusion assays in which exponentially growing cells were spread on NYG agar plates containing different concentrations of SFN (0 to 100 μM) and 20 μl of H₂O₂ (60 mM) in a central hole. After 3 days of incubation, the inhibitory zone diameters (a) of the cultures were measured and compared (b). Experiments were performed three times with similar results. Each dot represents the inhibitory zone diameter of a single biological replicate (n = 5). ***p < 0.001 (two‐way analysis of variance followed by Tukey's multiple comparisons test).

FIGURE 6

Identification of OxyR as a target of sulforaphane (SFN) that mediates the H₂O₂ resistance of Xanthomonas campestris pv. campestris XC1. (a and b) OxyR is responsible for the SFN‐mediated H₂O₂ sensitivity of XC1. The H₂O₂ (60 mM) resistance of XC1 and ΔoxyR in SFN (100 μM)‐containing medium was examined by performing a diffusion assay. Experiments were performed three times with similar results. Each dot represents the inhibitory zone diameter of a single biological replicate (n = 10). ***p < 0.001 (two‐way analysis of variance [ANOVA] followed by Tukey's multiple comparisons test). (c) OxyR regulates antioxidative gene expression in XC1. XC1 and ΔoxyR were grown in NYG medium to an OD₆₀₀ of 1.0 then 1 mM H₂O₂ was added to the medium and the cells were incubated for 10 min. The expression of antioxidant genes (katG, katA, and ahpC) in XC1 and ΔoxyR was examined using reverse transcription‐quantitative PCR. The bars represent the mean ± SD (n = 3). 16S rRNA was used as the endogenous control, the expression level in the wild‐type strain was assigned a numerical value of 1 and transcript levels were normalized to the 16S rRNA level. Experiments were performed three times with similar results. The statistical analysis was performed using two‐way ANOVA followed by Tukey's multiple comparison test (***p < 0.001). (d) SFN directly binds to OxyR. Microscale thermophoresis (MST) was used to quantify the binding affinity between SFN and the OxyR protein. Proteins were incubated with SFN in label‐free standard capillaries to conduct the MST assay. The titres of SFN ranged from 0.0305 to 1000 μM. The RpoA‐GST fusion protein was used as a negative control and phosphate‐buffered saline (PBS) was used as a blank control. The solid curve shows the fit of the data points to the standard K _d fit function. K _d, dissociation constant.

FIGURE 7

Sulforaphane (SFN) inhibits the DNA‐binding ability of OxyR. Gel shift assay showing that OxyR directly binds to p‐katA (the promoter region of katA) and p‐katG (the promoter region of katG), and the formation of the complex is weakened by adding SFN (a and b). (c) The saxF promoter, an negative probe control, does not bind to the OxyR protein under SFN‐containing or SFN‐free conditions. Different concentrations of OxyR and SFN were added to reaction mixtures containing 20 ng of probe DNA, and the reaction mixtures were separated on polyacrylamide gels. The band intensities were quantified and analysed using the ImageJ program. Numbers below the complex and above the free DNA represent the relative intensity of the corresponding band with the levels in lane 2 set to 1.0. Experiments were performed three times with similar results.

FIGURE 8

Sulforaphane (SFN) inhibits the H₂O₂ resistance of Xanthomonas oryzae pv. oryzae (Xoo) PX099A and X. oryzae pv. oryzicola (Xoc) RS105 growing in medium. (a and b) SFN induces oxyR expression in Xoc and Xoo. Xoo and Xoc cells were incubated with 20 mM SFN for 6 h in induction medium and transcription levels of the indicated genes were determined using reverse transcription‐quantitative PCR. 16S rRNA was used as the endogenous control, the expression level in the wild type was assigned a numerical value of 1, and transcript levels were normalized to the 16S rRNA level. Experiments were performed three times with similar results. The bars represent the mean ± SD (n = 3). (c and d) SFN inhibits the H₂O₂ resistance of Xoo PXO99A and Xoc RS105. H₂O₂ tolerance was determined by performing diffusion assays in which exponentially growing cells were spread on NYG agar plates containing SFN at different concentrations and 20 μl of H₂O₂ (30 mM) in a central hole. Experiments were performed three times with similar results. Each dot represents the inhibitory zone diameter of a single biological replicate (n = 5). ***p < 0.001, **p < 0.05 (unpaired t test).

FIGURE 9

Molecular model of sulforaphane (SFN)‐mediated inhibition of antioxidant gene expression by targeting OxyR. (a) In the process of normal growth and oxidative stress, OxyR automatically negatively regulates its own expression by binding to its own promoter (Christman et al., ; Tao et al., 1991). When bacteria sense H₂O₂, the transcriptional regulatory activity of OxyR is activated, and the expression of antioxidant enzyme genes (katA and katG) is regulated by OxyR, which directly binds to their promoters. In this state, bacteria are fully able to adapt to oxidative stress (“fully charged”). (b) In an environment containing SFN, SFN does not increase OxyR transcription, the transcription of oxyR is increased as a result of SFN disrupting the autoregulation. In addition, SFN directly binds to OxyR, thus weakening its ability to bind antioxidant enzyme gene promoters. In this state, the expression of antioxidative enzyme genes is inhibited and bacteria lose their ability to adapt to oxidative stress (“battery low”).